RMC-6236

KRas G12D

RMC-6236 （5 天）抑制 AsPC-1 (K-Ras G12D) 细胞活力，IC50 为 1–10 μM[1]。
RAS驱动的癌症占人类癌症的30%。RMC-6236是一种RAS（ON）多选择性非共价抑制剂，可抑制典型RAS亚型的突变型和野生型变体的活性GTP结合状态，对上述未满足的医疗需求具有广泛的治疗潜力。RMC-6236在RAS成瘾细胞系中表现出强大的抗癌活性，特别是那些在KRAS密码子12处存在突变的细胞系。[2]

在KRASG12X异种移植物模型的小鼠临床试验中，口服RMC-6236在体内是耐受的，并推动了多种肿瘤类型的深度肿瘤消退。转化PK/疗效和PK/PD建模预测，在RAS驱动的肿瘤患者中，每日剂量为100mg和300mg将分别实现肿瘤控制和客观反应。与此一致，我们在这里分别描述了两名晚期KRASG12X肺腺癌和胰腺癌患者（每天300mg）的客观反应，证明了RMC-6236在正在进行的I/Ib期临床试验（NCT05379985）中的初始活性。[2]

RAS-RAF TR-FRET。[2]
通过时间分辨荧光能量转移（TR-FRET）在反应缓冲液（25 mmol/L HEPES NaOH pH 7.3，0.002%吐温20，0.1%牛血清白蛋白，100 mmol NaCl、5mmol/L MgCl2）。加入化合物或DMSO对照（1%v/v）并孵育1.5小时，然后在PerkinElmer Envision平板读数器上测量TR-FRET（在320 nm处激发，20μs延迟，100μs窗口，闪光间隔2000μs；在单独的通道中在665 nm和615 nm处发射）。FRET比率（665/615 nmol/L发射）用于计算%抑制率，如下：[1-（样品的FRET比率-阳性对照的平均FRET比率）/（DMSO对照的平均FRET比率-阳性控制的平均FRET比例）]×100%。

---

CypA结合亲和力（KD1）。[2]
在Biacore 8K仪器上通过表面等离子体共振（SPR）评估化合物对CypA的结合亲和力。AviTag-CypA固定在链霉抗生物素蛋白传感器芯片上，并在测定缓冲液（10 mmol/L HEPES NaOH pH 7.4，150 mmol/L NaCl，0.005%v/v表面活性剂P20，2%v/v DMSO）中使不同浓度的化合物流过芯片。使用稳态亲和力模型或1:1结合（动力学）模型拟合SPR传感图，以评估KD1与CypA的结合。

---

RAS结合亲和力（KD2）。[2]
在Biacore 8K仪器上通过SPR评估化合物结合的CypA对上述突变致癌RAS蛋白的结合亲和力。AviTag RAS[1–169]固定在链霉抗生物素蛋白传感器芯片上，在测定缓冲液（10 mmol/L HEPES NaOH pH 7.4，150 mmol/L NaCl，0.005%v/v表面活性剂P20，2%v/v DMSO，25μmol/L CypA）中使不同浓度的化合物流过芯片。使用稳态亲和力模型或1:1结合（动力学）模型拟合SPR传感图，以评估RAS结合的KD。

---

细胞ERK磷酸化的AlphaLISA和中尺度发现（MSD）分析。[2]
将NCI-H441、Capan-2、HPAC或等基因无RAS MEF细胞接种在经组织培养处理的384孔和96孔板中，并孵育过夜。第二天，使用Labcyte Echo 550或Tecan D300e数字分配器将细胞暴露于化合物或DMSO对照（0.1%v/v）的连续稀释液中指定的时间点。孵育后，细胞被裂解，并按照制造商的方案，使用AlphaLISA SureFire Ultra pERK1/2（T202/Y204）检测试剂盒或MSD多阵列检测系统磷酸化/总ERK1/2全细胞裂解试剂盒（K15107D）测定ERK磷酸化水平。使用具有标准AlphaLISA设置的PerkinElmer Envision或MSD的Meso QuickPlex SQ120阅读器检测信号。对于AlphaLISA，数据表示为DMSO处理对照的百分比：100-100×（pERKDMSO-pERK处理）/（pERKDMSO-pERKmedia）。将来自pERK1/2的MSD信号除以MSD信号，得到总ERK1/2。将该比率归一化为载体（pERK/总ERK的百分比=（pERK处理/总ERK-处理的比率）/（pERKDMSO/总ERKDMSO的比率）×100）。对于这两种测定，数据均绘制为log M[化合物]的函数，并采用S形浓度响应（可变斜率）模型拟合数据，以估算Prism 9中的抑制剂EC50。

PRISM筛查[2]
将RMC-6236以8点浓度加入384孔板中，稀释3倍，一式三份。然后用解冻的细胞系池接种这些可用于检测的平板。贴壁细胞池以每孔1250个细胞的速度铺板，而悬浮液和混合贴壁/悬浮液池则以每孔2000个细胞的方式铺板。将处理过的细胞孵育5天，然后裂解。在条形码扩增和检测之前，将裂解物板折叠在一起。

---

2D细胞增殖分析。[2]
将NCI-H441、Capan-2和HPAC细胞接种在组织培养处理的384或96孔板中，并孵育过夜。使用Labcyte Echo 550或Tecan D300e数字分配器将细胞暴露于化合物或DMSO对照（0.1%v/v）的连续稀释液中，并在37°C下孵育120小时。在复合处理时，用强力霉素对强力霉素诱导的细胞系进行复育。根据制造商的方案，用CellTiter Glo 2.0试剂测定细胞存活率。使用PerkinElmer Enspire的SpectraMax M5平板阅读器检测发光。将发光信号归一化为载体处理的孔[%载体=（发光处理/平均值（发光载体）×100]。将数据绘制为对数摩尔[抑制剂]的函数，并将4参数S形浓度响应模型拟合到数据中以计算EC50。通过将处理过的细胞计数归一化为其各自的未处理细胞计数来计算生长百分比。

RMC-6236 Formulation. [2]
For in vitro studies, RMC-6236 was resuspended in dimethyl sulfoxide (DMSO) and used at 10 mmol/L stock concentration. For use in in vivo studies, RMC-6236 was prepared using formulation of 10/20/10/60 (%v/v/v/v) DMSO/PEG 400/Solutol HS15/water. The same vehicle formulation was used for all control groups.

---

RMC-6236 Treatment. [2]
Tumor-bearing animals were randomized and assigned into groups (n = 1–10/group). The vehicle at 10 mL/kg or RMC-6236 at indicated doses was administered via oral gavage daily, and animals were treated for 28 days, or up to 90 days if PFS was being assessed. Animals were terminated early if the tumor burden reached a humane endpoint, or adverse effect was observed with body weight loss as a surrogate. For single-dose PKPD study, mice were randomized and assigned into groups (n = 3/dose/time point). A single dose of RMC-6236 was administered orally at either 3, 10, or 25 mg/kg. Blood and tissues, including the tumor, brain, colon, ear skin, and muscle, were harvested at indicated time points. Whole blood was collected in K2EDTA Microtainer tubes, incubated for 5 minutes, and snap-frozen in liquid nitrogen. The tissue was either fixed in 10% formalin or snap-frozen in liquid nitrogen for further analysis.[2]

---

Mouse Blood and Tissue Sample Bioanalysis. [2]
The whole blood, tumor, brain, colon, and ear skin concentrations of RMC-6236 were determined using liquid chromatography–tandem mass spectrometry (LC/MS-MS) methods. Tissue samples were homogenized with a 10 × volume of homogenization buffer [methanol/15 mmol/L PBS (1:2; v:v) or 15 mmol/L PBS with 10% methanol]. An aliquot of whole blood or homogenized tissue (10, 20, or 40 μL) was transferred to 96-well plates (or tubes) and quenched with a 10 × volume of acetonitrile or 20 × volume of acetonitrile/methanol (1:1; v/v) with 0.1% formic acid containing a cocktail of internal standards (IS). After thorough mixing and centrifugation, the supernatant was diluted with water or directly analyzed on a Sciex 5500 or Sciex 6500+ triple quadrupole mass spectrometer equipped with an ACQUITY or Shimadzu UPLC system. A Halo 90Å AQ-C18 2.7 μm (2.1 × 50 mm) or an ACQUITY UPLC BEH C18 or C4 1.7 μm (2.1 × 50 mm) column was used with gradient elution for compound separation. RMC-6236 and IS (verapamil, celecoxib, glyburide, dexamethasone, or terfenadine) were detected by positive electrospray ionization using multiple reaction monitoring (RMC-6236: m/z 811/779; verapamil: m/z 455/165; celecoxib: m/z 382/362; glyburide: m/z 494/169; dexamethasone: m/z 393/373; terfenadine: m/z 472/436). The lower limit of quantification was 1 ng/mL or 2 ng/mL for blood, tumor, and other tissue.

---

PK/PD Relationship. [2]
Concentrations of RMC-6236 in tumor or normal tissues and percentage of DUSP6 inhibition as compared with the vehicle control from individual animals were collected and analyzed post a single dose of RMC-6236 ranging from 0.3 to 100 mg/kg (Supplementary Table S6). A 3-parameter sigmoidal exposure–response model was fitted to the data in GraphPad Prism to derive EC50 and EC90 values.

---

PK/Efficacy and PK/PD Modeling. [2]
For PK modeling, whole blood PK data from single or repeat dose administration of 25 or 40 mg/kg RMC-6236 to NCI-H441 xenograft tumor-bearing mice were used (Supplementary Table S9). RMC-6236 blood PK was best described using a one-compartment model with first-order absorption and elimination. Because intravenous data were not included in the modeling, the model was parameterized in terms of apparent clearance (CL/F) and volume of distribution (V/F), where F is the oral bioavailability.

---

Specifically, to understand the responsiveness of tumors harboring diverse oncogenic Kras variants to RMC-6236 treatment, lung tumors were initiated in B6 mice using a barcoded lentivirus pool including vectors encoding oncogenic KRAS mutant (G12C, G12V, G12D, G12A, Q61H, or G13D) cDNAs (Lenti;KrasMUT;BC). Thirteen weeks post tumor initiation, mice were treated for 3 weeks with either: (i) vehicle (10% DMSO, 20% PEG400, 10% Solutol HS15, 60% water) po qd and 10 mg/kg isotype rat igg2a[2a3] ip biw; or (ii) RMC-6236 20 mg/kg po qd.

[1]. Use of sos1 inhibitors to treat malignancies with shp2 mutations. Patent WO2022060583A1.

[2]. Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers. Cancer Discov. 2024 Jun 3;14(6):994-1017.
[3]. Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy. Nature. 2024 May;629(8013):919-926.
[4]. Drugging RAS: Moving Beyond KRASG12C. Cancer Discov. 2023 Dec 12;13(12):OF7.
[5]. State-of-the-art and upcoming trends in RAS-directed therapies in gastrointestinal malignancies. Curr Opin Oncol. 2024 Jul 1;36(4):313-319.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). Significance: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.[1]

---

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).[2]

---

Preliminary results from phase I trials respectively evaluating RMC-6236, a pan-RAS inhibitor, and HRS-4642, a KRASG12D inhibitor, indicate that both are safe and show promising signs of antitumor activity. These are just two of the candidate RAS therapies in a burgeoning development space as the field looks ahead to drugs that hit more than just KRASG12C.[3]

---

Purpose of review: Overall, the review underscores the evolving landscape of KRAS-targeted therapy and the potential for these approaches to improve outcomes for patients with gastrointestinal malignancies. It highlights the importance of ongoing research and clinical trials in advancing precision medicine strategies for KRAS-driven cancers. This review provides a comprehensive overview of the RAS signaling pathway and its significance in gastrointestinal malignancies. Recent findings: The introduction of KRAS inhibitor represents a significant advancement in the treatment landscape for KRAS-mutant cancers. In this review, we discuss upcoming trends in KRAS-targeted therapy, including the development of mutant-specific direct KRAS inhibitors like MRTX1133 and pan-RAS inhibitors such as RMC-6236. It also explores indirect RAS inhibitors targeting upstream and downstream components of the RAS pathway. Additionally, the review examines other upcoming strategies like combination therapies, such as CDK4/6 and ERK MAPK inhibitors, as well as adoptive cell therapy and cancer vaccines targeting KRAS-mutant cancers. Summary: Targeting RAS has become an important strategy in treating gastrointestinal cancer. These findings in this review underscore the importance of a multidisciplinary approach, integrating advances in molecular profiling, targeted therapy, immunotherapy, and clinical research to optimize treatment strategies for patients with KRAS-mutant gastrointestinal malignancies.[4]

C44H58N8O5S

811.06

810.43

C, 65.16; H, 7.21; N, 13.82; O, 9.86; S, 3.95

2765081-21-6

2765081-21-6; 2765091-21-0 (racemate)

164726578

White to off-white solid

5.1

162Ų

2

11

7

58

1470

5

FVICRBSEYSHKFY-UHFFFAOYSA-N

InChI=1S/C44H58N8O5S/c1-8-51-37-12-11-28-19-31(37)33(40(51)32-20-29(23-45-39(32)27(3)56-7)50-16-14-49(6)15-17-50)22-44(4,5)25-57-43(55)34-10-9-13-52(48-34)42(54)35(21-38-46-36(28)24-58-38)47-41(53)30-18-26(30)2/h11-12,19-20,23-24,26-27,30,34-35,48H,8-10,13-18,21-22,25H2,1-7H3,(H,47,53)

N-[21-ethyl-20-[2-(1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7-yl]-2-methylcyclopropane-1-carboxamide

RAS-IN-2; RAS In 2; RMC-6236; RMC 6236; RMC6236; EX-A6631; DA-77354; RMC-6236; Compound A122; RAS Inhibitor A122; (Z)-N-(11-ethyl-12-(2-(1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-61,62,63,64,65,66-hexahydro-11H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-2-methylcyclopropane-1-carboxamide; RMC6236

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~250 mg/mL (~308.2 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。